The invention relates generally to a method of enhancing expression of MHC class I molecules bearing endogenous peptides on the surface of a target cell and to a method of augmenting the immune response of a mammal to a tumor cell expressing low or nondetectable levels of MHC class I molecules bearing endogenous peptides. Further, the invention relates to a method of preparing tumor specific T cells which are therapeutically active against tumors.
The cytotoxic T lymphocyte (CTL) response is a major component of the immune system, active in immune surveillance and destruction of infected or malignant cells and invading organisms expressing foreign antigens on their surface. The ligand of the antigen-specific T cell receptor is a complex made up of a peptide fragment of a foreign antigen bound to major histocompatibility complex (MHC) molecules. In particular, cytotoxic T lymphocytes recognise peptide bound to MHC Class I molecules.
MHC class I molecules are normally expressed at the cell surface as ternary complexes formed by a heavy chain of 46 kD, a light chain called xcex22-microglobulin (xcex22m) of 12 Kd and a peptide composed of 8-10 amino-acids (van Bleek, G. M. and S. G. Nathenson, Nature 348:213, 1990; Zhang, W. et al., Proc. Natl. Acad. Sci. USA 89:8403, 1992; Matsumura, M. et al., Science 257:927, 1992; and Latron, F., et al., Science 257:964, 1992). Formation of the ternary complex is thought to involve transport into the lumen of the endoplasmic reticulum (ER) of peptides generated by protein degradation in the cytoplasm (Nuchtern, J. G. et al., Nature 339:223, 1989;Yewdell, J. W. and J. R. Bennink, Science 244:1072, 1989; and Cox,
J. H. et al., Science 247:715, 1990). The study of mutant cell lines selected for their low expression of MHC class I molecules at the cell surface has provided insights into the molecular events required for antigen processing. These studies have allowed the identification of two genes located in the MHC region which encode proteins of the ATP binding cassette (ABC) family. These genes, called TAP-1 and TAP-2, have been implicated in transport of peptides from the cytoplasm to the lumen of the ER (Deverson, E. V. et al., Nature 348:738, 1990; Trowsdale, J. et al., Nature 348:741, 1990; Spies, T. et al., Nature 348:744, 1990; Monaco, J. J. et al., Science 250:1723, 1990; Spies, T. and R. DeMars, Nature 351:323, 1991; Bahram, S. et al., Proc. Natl. Acad. Sci. USA 88:1009.4, 1991; Spies, T. et al., Nature 355:644, 1992; Kelly, A. et al., Nature 355:641, 1992; Powis, S. H. et al., Proc. Natl. Acad. Sci. USA 89:1463, 1992; and Colonna, M. et al., Proc. Natl. Acad. Sci. USA 89:3932, 1992). Two other MHC linked genes, LMP-2 and -7 (Monaco, J. J. and McDevitt, 1982, Proc. Natl. Acad. Sci. USA 79:3001), are components of the proteasome, a cytoplasmic multicatalytic protease complex, which is likely responsible for some aspects of protein degradation for antigen processing (Ortiz-Navarette, V. et al., Nature 353:662, 1991; Brown, M. G. et al., Nature 353:355, 1991; Glynne, R. et al., Nature 353:357, 1991; Martinez, C. K. and J. J. Monaco, Nature 353:664, 1991; Kelly, A. et al., Nature 353:667, 1991; Yang, Y. et al., Proc. Natl. Acad. Sci. USA 89:4928, 1992; Goldberg, A. L. and K. L. Rock, Nature 357:375, 1992).
The mouse mutant lymphoma cell line RMA-S expresses low levels of class I molecules at the cell surface compared to the wild type RMA cells (Ljunggren, H.-G. et al., J. Immunol. 142:2911, 1989; and Townsend, A. et al., Nature 340:443, 1989). Influenza virus infected RMA-S cells present influenza peptides in the context of Db molecules inefficiently and are only weakly recognized by specific CTL (Townsend, A. et al., Nature 340:443, 1989). Transfection with the putative transporter gene, TAP-2, complements this deficiency (Powis, S. J. et al., Nature 354:528, 1991; and Attaya, M. et al., Nature 355:647, 1992). The endogenous TAP-2 gene of RMA-S cells was shown to contain a point mutation which introduces a stop translation codon resulting in an incomplete and defective TAP-2 protein (Yang, Y. et al., J. Biol. Chem. 267:11669, 1992). Despite the defective TAP-2 protein in RMA-S cells, antigenic peptides from vesicular stomatitis virus (VSV) bypass the defect and are presented to specific CTL by Kb molecules in RMA-S cells (Esquivel, F., et al., J. Exp. Med. 175:163, 1992; and Hosken, N. A. and M. J. Bevan, J. Exp. Med. 175:719, 1992). The VSV-nucleocapsid (N) peptide, VSV-N 52-59, has been shown to be the major peptide presented by Kb molecules on VSV infected cells (van Bleek, G. M. and S. G. Nathenson, Nature 348:213, 1990). The presence of the wild-type TAP-1 protein in RMA-S cells may be sufficient for translocation of the VSV-N 52-59 peptide to the ER lumen (Powis, S. J. et al., Nature 354:528, 1991; Attaya, M. et al., Nature 355:647, 1992; and Yang, Y. et al., J. Biol. Chem. 267:11669, 1992). Alternatively, the VSV-N 52-59 peptide may not need a functional transporter for transport into the lumen of the ER. Expression of minigene-encoded viral peptide epitopes in T2 cells (Zweerink, H. J. et al., J. Immunol. 150:1763, 1993) and in-vitro translation and translocation using microsomes from T2 cells (Levy, F. et al., Cell 67:265, 1991) support this contention.
A separate class of antigen processing variants are those in which the assembly and the surface expression of MHC class I molecules are entirely inducible by IFN-xcex3 (Klar, D. and G. J. Hxc3xa4mmerling, EMBO J. 8:475, 1989). For example in the small lung carcinoma cell line, CMT.64, recognition by influenza virus specific CTL does not take place unless induced with IFN-xcex3 (Sibille, C. et al., Eur. J. Immunol. 22:433, 1992). The very low amount of all proteasome components present in uninduced CMT.64 cells is presumed to be responsible for their phenotype (Ortiz-Navarette, V. et al., Nature 353:662, 1991). Exogenous influenza peptides can bind to Db molecules on CMT.64 cells and complement recognition by influenza specific CTL (Sibille, C. et al., Eur. J. Immunol. 22:433, 1992). In addition, it has been found that the xcex22m and the VSV-N 52-59 peptides added exogenously to these cells complement recognition by VSV specific CTL restricted to Kb (Jefferies W. A. et al., 1993, J. Immunol. 151:2974). The amount of xcex22m and of heavy chains synthesized in these cells may limit the amount of MHC class I expression on the cell surface (Jefferies et al, supra, 1993). A dysfunction of the putative peptide transporters and/or in the generation of the peptide may be responsible for the CMT.64 phenotype which may represent a mechanism to downregulate MHC class I expression, a feature common to many carcinomas.
Restifo, N. R. et al. (J. Exp. Med. 177:265-272, 1993) studied the antigen processing efficiency of 26 different human tumor lines using a recombinant vaccinia virus (Vac) to transiently express the Kd molecule. Three cell lines, all human small cell lung carcinoma, consistently failed to process endogenously synthesized proteins for presentation to Kd-restricted, Vac-specific T cells. Pulse-chase experiments showed that MHC class I molecules were not transported by the cell lines from the endoplasmic reticulum (ER) to the cell surface. Northern blot analysis of the cells revealed low to nondetectable levels of mRNAs for MHC-encoded proteasome components LMP-7 and LMP-2 as well as the putative peptide transporters TAP-1 and TAP-2.
The present inventors have surprisingly found that, despite the multiple antigen processing deficiencies in CMT.64 cells, TAP-1 transfected into the cells, alone was sufficient to induce CTL recognition of VSV infected cells in the absence of IFN-xcex3 induction. Importantly, TAP-1 was shown to function independently of TAP-2 in peptide transport, as the transfected cells remained negative for TAP-2 expression. It was also demonstrated that TAP-1 alone delivered specific peptides to the site of MHC assembly, permitting stable complexes to form with resultant transport and expression at the cell surface to permit immune surveillance by cytotoxic T lymphocytes (CTL).
Further, it was demonstrated that some peptides are translocated in the ER by TAP-2 alone. For example it was shown that TAP-2 alone in the absence of TAP-1, is sufficient to enhance processing and presentation of the influenza NP366-374 peptide.
The present inventors have importantly shown that mice injected with high loads of CMT.64 cells transfected with TAP-1 and TAP-2 show increased survival rates and significantly decreased pathology and metastasis compared to mice injected with wild type CMT.64 cells.
The invention therefore contemplates a method for enhancing expression of MHC class I molecules bearing endogenous peptides on the surface of a target cell expressing low or nondetectable levels of MHC class I molecules and expressing low or nondetectable levels of TAP-1 and TAP-2 transporter proteins comprising: introducing into the target cell a nucleic acid molecule comprising a sequence encoding TAP-1 or TAP-2 under control of a suitable promoter, and expressing TAP-1 or TAP-2 in the target cell under suitable conditions thereby enhancing processing and presentation of MHC class I molecules bearing endogenous peptides. Preferably, the target cell is a tumor cell which, additionally has a deficiency in proteasome components, most preferably a small lung cell carcinoma cell. In one embodiment, the processing and presentation of endogenous peptides which have the motif RGYVYQGL (SEQ ID NO: 1), which is restricted to Kb are enhanced by introducing a nucleic acid molecule encoding TAP-1. In a second embodiment, the processing and presentation of endogenous peptides which have the motif ASNENMETM (SEQ ID NO:2), which binds to H-2Db, are enhanced by introducing a nucleic acid molecule encoding TAP-2.
In an embodiment of the method, a nucleic acid molecule comprising a sequence encoding TAP-1 or TAP-2 and a second nucleic acid molecule comprising a sequence encoding an antigenic peptide, preferably a T-cell receptor interactive antigen, under control of a suitable promoter are introduced into the target cell.
The invention also relates to the use of a recombinant viral vector comprising a nucleotide sequence encoding TAP-1 or TAP-2 and a nucleotide sequence encoding an antigenic peptide vector to enhance cell surface expression of an MHC class I molecule bearing endogenous peptides in a tumor cell expressing low or nondetectable levels of MHC class I molecules and expressing low or nondetectable levels of the transporter proteins TAP-1 and TAP-2.
The invention still further provides a method of augmenting the immune response of a mammal to a tumor cell expressing low or nondetectable levels of MHC class I molecules bearing endogenous peptides comprising: introducing a nucleic acid molecule comprising a sequence encoding TAP-1 and/or TAP-2 into the tumor cell under control of a suitable promoter and; expressing TAP-1 and/or TAP-2 in the tumor cell under suitable conditions, thereby enhancing processing and presentation of MHC class I molecules bearing endogenous peptides permitting recognition by the mammal""s immune response, particularly recognition by cytolytic T cells.
In an embodiment of the method, the nucleic acid molecule is introduced into the tumor cell in a vaccinia virus. A preferred embodiment comprises introducing an additional nucleic acid molecule into the tumor cell; the additional nucleic acid molecule comprising a sequence encoding an antigenic peptide under control of a suitable promoter and; expressing the antigenic peptide in the tumor cell under suitable conditions, thereby enhancing processing and presentation of MHC class I molecules bearing the antigenic peptide permitting recognition by the mammal""s immune response, most preferably, the cytolytic T lymphocyte response. In a particular embodiment, the virus may be used as a vaccine to enhance the immune response to the antigenic peptide.
The invention still further relates to a method of preparing tumor specific T cells which have anti-tumor properties comprising removing tumor cells from a subject; introducing a nucleic acid molecule encoding TAP-1 or TAP-2 under the control of a suitable promoter into the tumor cells; implanting the tumor cells in the subject or a mammal have a reconstituted immune system of the subject; and harvesting tumor specific T cells. The tumor specific T cells may be used as a therapeutic agent in vivo in the subject.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, reference is made herein to various publications, which are hereby incorporated by reference in their entirety.